Lab Test 1

Question 1

Understand all laboratory safety information and policies listed

Answer 1

As you perform the lab exercises, always follow experiment procedures and instructions for cleanup and storage. Laboratory kits contain the following potential hazards:

Items in your lab kit (see Figure 1) can be especially dangerous to children and pets. Always conduct experiments away from children and pets and safely store equipment out of their reach.

Your lab kit may contain chemicals and materials that can cause burns if mishandled, and serious illness and/or death if consumed. Never consume materials used in the experiment. Follow experiment procedures carefully.

Your lab kit may contain small items and materials that could cause choking, injury, or death.

Many items are made of glass and/or have sharp edges that pose potential risks for cuts and scratches.

Question 2

Personal safety precautions

Answer 2

1. Safety goggles must be worn for the duration of any experiments that require safety goggles. Goggles will provide protection against chemical spills and splashes, as well as projected objects or debris.
2. Eating, drinking, or smoking should never occur near the work space while performing laboratory procedures. Do not use any lab equipment as containers for food or beverages.
3. Long hair must be tied back, clothing must fully cover the arms and legs, and closed-toed shoes must be worn when performing experiments.
4. Work areas must be kept clean and tidy at all times. Only laboratory instructions, worksheets, and/or reports should be brought to the work area. Other materials (e.g., books, purses, backpacks) must be stored in another area.
5. Work surfaces for performing experiments must be solid, non-porous, heat tolerant surfaces, such as metal, stone, or laminate. Do not use plastic, wood, or glass surfaces.
6. Work areas should always be well-ventilated.
7. A physician should be consulted prior to conducting any experiments if you are pregnant, allergic to certain chemicals, or immunocompromised in any way.
8. Caution should be practiced when performing experiments that require physical activity. While the physical activities in our lessons are not necessarily dangerous, you should always practice caution when completing them. If a procedure requires vigorous exercise, consult a physician. If you are unable to complete the task safely, ask a partner to participate in your place.

Question 3

Understand laboratory safety behavior

Answer 3

1. read labels of all chemicals
2. remove all jewelry and loose clothing
3. tie back hair
4. wash hands before and after each experiment
5. before experiment: gather all equipment
6. workspace should be well ventilated area clear of clutter
7. do not chew gum or put anything in your mouth during the experiment
8. work area should be nonporous and heat tolerant substance (metal, stone, or laminate)
9. keep cleanup materials nearby
10. always use proper wafting techniques
11. if your experiment uses an open flame or heating of any kind, remember to use a hot pad or towel when touching or move hot glassware or equipment
12. as you perform your experiment, remain aware of your surroundings
13. never leave your experiments open and unattended
14. select a safe space away from children and pets
15. during cleanup dispose of chemicals, used pipettes, consumable materials, or dissections properly
16. after you have finished your experiment, thoroughly clean your work surface with a 10% bleach solution
17. properly remove PPE and wash hands

Question 4

What is correct PPE?

Answer 4

Personal Protective Equipment: apron, gloves, goggles, face mask

To better protect the body from chemical spills, wear the following:

Long-sleeved shirts
Full-length pants
Closed-toe shoes
Protective nitrile gloves and a face mask (as directed)
Be sure to pull or pin hair back to avoid possible contact with flames, chemicals, or any other experiment components.

Question 5

Understand proper clean-up and handling procedures for all examples given

Answer 5

Biological Microorganisms
Culture Spills

If any culture material is spilled, spray the spill with a disinfectant, such as a bleach-based cleaner, and allow it to sit for 15 minutes. Clean the spill with paper towels, then discard them in the garbage, and repeat the cleaning process.

Culture Clean-up

When finished with a culture, add pure bleach to the plate or tube and allow it to sit for 2 hours. Tape the lid or cap shut, wrap in a paper towel, place in a sealable plastic bag, and discard the materials in the garbage.

Contaminated Equipment
After using loops, swabs, toothpicks, spreaders, or any other equipment to transfer cultures, immerse the contaminated equipment in a small container of pure bleach for at least 2 hours. Wrap all bleached items in a paper towel, place in a sealable plastic bag, and discard in the garbage. Or, if the item is to be reused in other experiments, thoroughly wash the item with soap and warm water after applying bleach for 2 hours.

This figure shows a scientist is holding up a petri dish with bacteria on it.
Image copyright Michal Kowalski, 2014. Used under license from Shutterstock.com.
Chemicals
Acid Splatter

When water is added to concentrated acid, the water may cause the acid to splash out of the container. Splattering is less likely to occur if you add acid slowly to the water. Remember this AAA rule: Always Add Acid to water, never add water to acid.

Chemical Ingestion

Virtually all chemicals found in a laboratory are potentially toxic. To avoid ingesting dangerous chemicals, never taste, eat, or drink anything while in the laboratory. All laboratories, and especially those in home kitchens, should always be thoroughly cleaned after experimentation to avoid this hazard. In the event of any chemical ingestion, immediately call the National Poison Control Center and consult a physician.

Chemical Spills

Flesh burns may result if acids, bases, or other caustic chemicals are spilled and come in contact with skin. Flush the exposed skin with a gentle flow of water for several minutes at a sink or safety shower. Neutralize acid spills with sodium bicarbonate (baking soda). If eye contact is involved, use the eyewash station or a comparable substitute. Use the spill containment kit until the spill is neutralized.

Heating Chemicals

Heat solid and liquid chemicals with great care to prevent explosions and accidents. Never leave an ignited heat source unattended.

Heating Liquids in Beakers or Flasks: Ensure that the containers are well supported above the heat source. Typically, the containers are placed on a stand made of wire gauze and iron, and then the heat source is placed under the stand below the container.
Note: Graduated cylinders, volumetric flasks, and other pieces of glassware are not designed to be heated. Always ensure you are using heatproof glass before applying it to a heat source.

Heating Liquids in Test Tubes: Always use a test tube rack or holder and never point a heated test tube towards anyone or yourself. Evenly heat the test tube contents by carefully moving the test tube back and forth through the flame. Heat the test tube near the top of the liquid first, as heating from the bottom of the test tube may cause the liquid to boil and eject from the tube.

Glassware and Tubing
Glass Tubing Hazards

Never force a piece of glass tubing into a stopper hole. The glass may snap, and the jagged edges can cause serious injury. Before inserting glass tubing into a rubber or cork stopper hole, be sure the hole is the proper size. Lubricate the end of the glass tubing if instructed to do so in the procedures. While grasping the tubing with a heavy glove or towel, gently but firmly twist the tubing into the hole. Treat any cuts or scrapes with appropriate first aid.

Heated Test Tube Splatter

Splattering and eruptions can occur when solutions are heated in a test tube. To minimize this danger, direct the flame toward the top rather than the bottom of the test tube. Gently agitate the tube over the flame to heat the contents evenly. You should never point a heated test tube towards anyone or yourself.

Shattered Glassware

Take special caution when working with any type of laboratory glassware to prevent shattering or breaking that can result in injuries. Only heat glassware that is heatproof and always ensure glassware is placed on a stable surface.

Question 6

Cleaning and Disposal Instructions for Common Materials

Answer 6

Instrument Use

• Do not return chemicals to their containers after they have been dispensed.
• Examine glassware and labware before each experiment. Ensure all labware is clean prior to each experiment. Never use chipped or cracked glassware or broken labware.

Cleaning Instructions

• Use a soft cloth or test tube cleaning brush, mild dishwashing detergent, and warm water to loosen solids or oils from glassware, plastics, and other laboratory equipment after completing an experiment. Thoroughly rinse the items with distilled water and allow them to air dry on clean paper towels or a clean dish towel.
• Return all clean, dry equipment to your kit for future use.

Disposal

• Contact your local waste management agency to obtain the protocol for the safe disposal of waste before discarding any chemical reagents or dissection specimens.
• To properly remove your gloves, first remove one glove by pulling at the fingers until the glove is removed. Hold the removed glove in the still gloved hand. Then, reach under the base of the second glove and pull the glove off, turning it inside out as you remove it so that it fully encloses the first glove. Dispose of your gloves by putting them in the trash.
• Wrap non-chemical experiment items in newspaper or paper towels and dispose of them into the household garbage after the completion of the course. Ensure all garbage is housed in a securely covered trash container that cannot be accessed by children or animals

Question 7

Understand how to select the best location for your at home laboratory

Answer 7

The best place to perform experiments at home will vary depending on the nature of each experiment. However, a home laboratory has the following characteristics:

• Located in an uncluttered room away from children and pets.
  If your experiment requires an extended period of time to be completed, make sure it is performed in a safe place away from others in your home.
• Located in a well-ventilated area. Rooms that have a window or door that can be opened for fresh air, ventilation, and fume exhaust are great choices.
• Located near a source of running water. Sinks and showers are ideal for lab clean up and in case of chemical spills.
• Has a solid, non-porous, heat tolerant surface such as metal, stone, or laminate to work on. Do not use a plastic, wood, or glass surface.
• Has access to safety equipment in your home such as showers, fire extinguishers, and sinks.

Question 8

Understand types of injuries and how to treat

Answer 8

First Aid Kit
A kit of basic first aid supplies is used for the emergency treatment of injuries and should be standard in both formal and informal laboratories. A first aid kit should always be well-stocked and easily accessible. First aid kits are readily available online and at grocery stores and pharmacies.

Eyewash Station
An eyewash station is used if your eyes are exposed to a microorganism or if a harmful chemical is splashed into your eyes or face. In the home, use the nearest sink and flush your eyes or face with cool water for at least 20 minutes. If the sink has a sprayer, use it to rinse your eyes or face while making sure the water drains into the sink.

Safety Shower
Safety showers are used to remediate exposure to large quantities of microbes or for large chemical spills. Safety showers are also used if a hazardous chemical is spilled on a person and that person is unable to rinse it off thoroughly in a sink. They may even be used in the event that clothes or an object catches fire. A standard home bathroom shower sufficiently serves as a safety shower should the need arise. Stand under the flow of water while wearing the contaminated clothing for at least 20 minutes.

Question 9

Explore Chemical Handling Hazards

Answer 9

The labels on chemical containers indicate any hazards involved in shipping and handling the compound. The four-diamond system and Globally Harmonized System (GHS) of pictograms are the most common systems for identifying hazardous materials. Both systems can appear on Safety Data Sheets (SDS).

The four-diamond symbol was developed by the National Fire Protection Association and uses a color-coded symbol with four diamonds. These colored diamonds provide the following information about the the hazards associated with chemical handling:

• Fire hazard (top, red diamond)
• Health Hazard (left, blue diamond)
• Specific Hazard (bottom, white diamond, e.g., radioactivity)
• Reactivity Hazard (right, yellow diamond)
  Select the diamonds for further explanations of each hazard.

Question 10

Understand Safety Data Sheets, what they contain and how to find them

Answer 10

An SDS is designed to provide chemical, physical, health, and safety information about chemical reagents and supplies. The SDS provides users and emergency personnel with the proper procedures for how to handle, store, transport, use, and dispose of chemicals in a safe manner.

While there is no standard format for an SDS, any SDS provides basic information about physical data, toxicity, health effects, first aid procedures, chemical reactivity, safe storage, safe disposal, required protective equipment, and spill cleanup procedures.

An SDS is required to be readily available at any business where any type of chemical is used. Even daycare centers and grocery stores need SDSs for their cleaning supplies. Thus, SDSs are also a core safety feature for campus science laboratories.

SDS information for Science Interactive materials can be found in the following ways:

Finding the SDS on our website.
Searching “Safety Data Sheet” and the name of the chemical on the Internet.
Requesting more information from your instructor.

Question 11

Understand how to pour agar plates

Answer 11

Clean work area
write NA and info on plates
Boil agar (water needs to be 1mm above agar line so it heats through consistently
Tip lids so it won’t get condensation
check every now and then
when ready take out and put in cup of hot water
lift lid barely up and pour in plate
let cool with lid barely up
store plates inverted so condensation does not ruin plates

Question 12

Understand the helpful hints given of introduction to Microbiology

Answer 12

1. You will be incubating cultures for many of the experiments. Locate a warm area, 21°C-25°C, in your home away from drafts and direct light. An empty cabinet is ideal. If cultures must be incubated on a counter or table, make sure to cover with a box and keep secure from children and pets.
2. There are 30 pairs of safety gloves included in the Microbiology kit, allotting two pairs per experiment and 8 extra pairs to be used as necessary. If you require additional nitrile gloves, they may be purchased from a variety of stores and online retailers.
3. Always review the time allocations before beginning a laboratory to ensure that you are managing your time appropriately.
4. Always use aseptic (sterile) technique as instructed in the procedural steps.
5. Always follow safety rules and wear gloves, goggles, and a face mask when working with bacteria.
6. At the end of each exercise, read closely as to whether you should save the samples for future experiments or bleach and dispose of the samples.
7. When beginning an experiment, always wipe down your workspace with a 10% bleach solution or bleach-based cleaning product.
   After completing an experiment, always wipe down your workspace with a 10% bleach solution or bleach-based cleaning product.

Question 13

Understand Addie Rerecich’s case including the bacteria, cause, treatment and outlook

Answer 13

Bacteria: Infected mostly with community MRSA (staph infections resistant to many antibiotics)
o She then was infected with Stenotrophomonas from the ECMO

Cause: MRSA from playground and cut on knee
Stenotrophomonas from the ECMO

Treatment: On antibiotics, bp dropping, next morning she needed oxygen with a mask, infected boils, then had to go on ECMO
After the lung transplant they finally were able to bring Addie home

Outlook:
Follow up 2 years later
o Had to bring monitors and learn basic movements again
o Has to take handful of pills 2 times a day
o Has pneumonia 5 times in the last 2 years
o Have to be very careful with bacteria
o Risks of dying just keep going up and up
o Addie never got better, they just bought some time

INFO

Cause:
* Tuscon Arizona May 2011
o Addie
o Dangerous bacteria that is increasingly resistant to antibiotics
o Started with a pain in her hip
o Symptoms of a virus
o Pain spread and fever got worse
o On antibiotics, bp dropping, next morning she needed oxygen with a mask, infected boils, then had to go on ECMO
o Infected mostly with community MRSA (staph infections resistant to many antibiotics)
o She then was infected with Stenotrophomonas from the ECMO
o Gram negative bacteria = body armor around bacteria
o Then the Stenatrophomonus become pan resistant
o After the lung transplant they finally were able to bring Addie home
o Follow up 2 years later
o Had to bring monitors and learn basic movements again
o Has to take handful of pills 2 times a day
o Has pneumonia 5 times in the last 2 years
o Have to be very careful with bacteria
o Risks of dying just keep going up and up
o Addie never got better, they just bought some time

Question 14

Understand David Ricci’s case including the bacteria, cause, treatment and outlook

Answer 14

Bacteria: India ‘Super bug’ – B-lactamase 1 (NDM-1) (resistance gene that can turn bacteria into superbugs)
 In hospitals and in the environment

Cause: Leg ran over by a train in India and then become infected in the hospital

Treatment: in India, the doctors just kept having surgeries to scrape at his leg
David went back to Washington and brought NDM-1 to the US
 Had to go back to 1940s colistin which was toxic to the body
o Had to cut off more of David’s leg
o After 3 more surgeries and highly toxic rounds of antibiotics, the doctors believe they got rid of all the NDM-1

Outlook: They believe they got rid of all the NDM-1 but Ricki always worries

INFO

David Ricci– 19yr old in Kolkuta India
o Leg ran over by a train
 Had to cut leg off
o Went to another doctor and there were no more complications
o They wanted him to have more surgeries
o India ‘Super bug’ – B-lactamase 1 (NDM-1) (resistance gene that can turn bacteria into superbugs)
 In hospitals and in the environment
o David went back to Washington and brought NDM-1 to the US
 Had to go back to 1940s colistin which was toxic to the body
o Had to cut off more of David’s leg
o After 3 more surgeries and highly toxic rounds of antibiotics, the doctors believe they got rid of all the NDM-1

Question 15

Understand what happened at the NIH in Maryland including all the details introduced to you

Answer 15

• New York summer 2011
  o KPC
   Lives in digestive system and a gene that can spread it’s resistant to other bacteria
  o Patient in NYC transported to Bethesda MD
  o So many aseptic techniques
   Enhanced contact isolation – have to wear gloves and gowns
  o 5 weeks after NY patient recovered a KPC bacteria was in a respiratory culture
   Male 34 yrs old
   First thought it was a second infection
   More patients started getting KPC
   Tried oral to IV antibiotics
   Tried like 5 to 6 at a time
   Tried an exmperimental antibiotics
  o Aseptic techniques
   Moved to a separate KPC ICU
   Used robots to clean
   But it was still spreading
  o How it spread
   Some can be silent carriers
   The outbreak spread past the ICU
   Patients started to die
  o 6 months the outbreak finally subsided
   18 infected and 6 died from it
  o KPC never gone from NIH now
  o Found in hospitals in about 44 states

Question 16

Understand why antibiotics become less resistant to the level discussed in the video

Answer 16

o The more we use, the more rapidly we lose
o The more you expose a bacteria to a antibiotic, then the more the bacteria become resistant
o Up to ½ of antiobiotic use in ineffective or inappropriate

Question 17

Understand why there are not a lot of new antibiotics being produced

Answer 17

o Most pharmacys were pulling out of antibiotics except for Pfizer
o Pfizer brought in John Quinn
 His team started creating several different compounds to breach gram negatives
o Shifted to vaccines
o Antibiotic pipeline is drying up
o Questions are now turning to see if the gov. is going to help out
o Antimicrobial resistance is down to #70 on the governments list to do basic research

Question 18

Understand how many infections there are and if that is accurate or not

Answer 18

o CDC now decided to shoutout about nightmare bug
o We don’t have a comprehensive plans or know the answers of the data
o At least 2 million are infected and about 23,000 die from resistant bacteria
can’t tell if some may be asymptomatic

Question 19

Know vocabulary listed for Microorganisms, Aseptic Technique and Cultures

Answer 19

ASEPTIC TECHNIQUES - the techniques needed to prevent the accidental or inadvertent spread of microorganisms beyond the intended working environment.

BROTH - liquid media

AGAR - * Solid media is prepared as either slants or plates. See Figures 3 and 4. The most common solidifying agent in both slants and plates is agar. Agar does not melt until it reaches a temperature of about 80°C (176°F). Conversely, once it has melted, it can be cooled to about 45°C (113°F) before it solidifies. In addition, agar has the ability to grow microorganisms over a large temperature range.

SLANTS - * Slants are primarily used for storage and transport of microorganisms. See Figure 3. Organisms stabbed into the media are protected from desiccation, allowing them to survive much longer. Slants can be stored for weeks, even months, without significant death to the organisms that they house. The screw cap and the small size of the vial allow a scientist to easily transport a culture from one place to another.

PLATES - * Plates provide a larger surface area for culturing microbes than slants. See Figure 4. Plates are commonly used for isolating microbes.

MIXED CULTURES - Culture samples taken from individuals or the environment contain many types of microbes.

CONTAMINATED CULTURE - occurs when unknown microorganisms are inadvertently introduced often via poor technique

GENERAL PURPOSE MEDIA - These media contain a rich variety of nutrients that will facilitate the growth of a wide range or microorganisms and is therefore ideal for generalized growth

NUTRIENT AGAR PLATE - which will be used during this laboratory. In future laboratories, more specialized media that select for specific types of microorganisms will be introduced which can aid in microbe species identification.

STREAK PLATE - primary mechanism for isolating bacteria. This technique utilizes a four quadrant dilution that systematically reduces bacterial numbers until they are diluted enough to form isolated colonies. The procedure is shown in Figure 5 and can be separated into four defined steps. Carefully read the following steps as you will be streaking a number

INOCULATION LOOP - . An instrument called an inoculation loop is sterilized and then placed flat in the center edge of the first quadrant. The inoculation loop is used to spread a small portion of the organism from the first quadrant into the second quadrant

MORPHOLOGY - size, shape, and other physical characteristics that can be used to identify microorganisms

Question 20

Understand aseptic technique

Answer 20

• Aseptic techniques are the techniques needed to prevent the accidental or inadvertent spread of microorganisms beyond the intended working environment.

Question 21

Understand how to perform aseptic technique and the steps

Answer 21

1. Hands must be washed, work surfaces must be cleared of unnecessary items, and sterilized with a 10% bleach solution or a bleach-based cleaner before beginning the Experimentation. See Figure 1.
2. The equipment used to isolate cultures has to be sterilized. Either an open flame or isopropyl alcohol may be used to eliminate contamination that is naturally picked up from the environment. In campus laboratories, equipment is often sterilized with an autoclave or micro-incinerator.
3. When opening cultures or sterile media:
   a. Work quickly and efficiently.
   b. Do not talk or breath over the culture.
   c. When tubes and vials are open, keep them tilted away from the mouth and nose.
   d. Never lay caps or lids on a bench or work area, as these areas most likely have contaminants.
   e. When working with a culture, ensure the instruments used are sterile. this will include inoculation loops and swabs. Sterile instruments are provided by Hands-On Labs, Inc. (HOL) You must work to maintain the instruments’ sterility
4. Some general precautions include the following:
   a. Avoid producing aerosols (anything that can be introduced into the air). This includes avoiding splashing when pipetting media and inoculating cultures.
   b. Keep the lid to the culture closed whenever possible.
   c. Incubate plates inverted (agar-side up). Agar contains a lot of water. When plates are incubated with the lid side up, water will condense on the lids; this condensation can drip back onto the agar plate and possibly contaminate and/or smear the results.
   d. Always write on the bottoms of the plates, not the lids. This will prevent unlabeled plates if lids are misplaced.
   e. When not in use, tubes and plates should be properly stored away from children and pets.
   f. All equipment and work surfaces should be cleaned with a 10% bleach solution or bleach based cleaning product at the end of the experiment.

Question 22

Understand the types of media and when it might be used

Answer 22

• HOL provides various forms of media that grow cultures of microbes. In general, media exist in two major forms: liquid and solid. One liquid type of media is broth, which is depicted in Figure 2. A microbiologist uses a broth primarily to increase the number of microorganisms.
• Solid media is prepared as either slants or plates. See Figures 3 and 4. The most common solidifying agent in both slants and plates is agar. Agar does not melt until it reaches a temperature of about 80°C (176°F). Conversely, once it has melted, it can be cooled to about 45°C (113°F) before it solidifies. In addition, agar has the ability to grow microorganisms over a large temperature range.
• Slants are primarily used for storage and transport of microorganisms. See Figure 3. Organisms stabbed into the media are protected from desiccation, allowing them to survive much longer. Slants can be stored for weeks, even months, without significant death to the organisms that they house. The screw cap and the small size of the vial allow a scientist to easily transport a culture from one place to another.
• Plates provide a larger surface area for culturing microbes than slants. See Figure 4. Plates are commonly used for isolating microbes. Culture samples taken from individuals or the environment contain many types of microbes. These mixed cultures must be separated into individual colonies of only one type of microbe to be successfully analyzed. A pure culture contains only one species of microorganism. Proper use of aseptic techniques prevents pure cultures from being contaminated. A contaminated culture occurs when unknown microorganisms are inadvertently introduced often via poor technique. In addition, bacterial colony morphology (the shape of a group of bacteria) can help distinguish among different species when using a plate.
• Secondary descriptive terms are often used when describing various media. Some media are described as a general purpose media. These media contain a rich variety of nutrients that will facilitate the growth of a wide range or microorganisms and is therefore ideal for generalized growth. One example of general purpose media is a Nutrient Agar plate or NA plate, which will be used during this laboratory. In future laboratories, more specialized media that select for specific types of microorganisms will be introduced which can aid in microbe species identification.

Question 23

Understand the different types of cultures

Answer 23

. Culture samples taken from individuals or the environment contain many types of microbes. These mixed cultures must be separated into individual colonies of only one type of microbe to be successfully analyzed.

A pure culture contains only one species of microorganism. Proper use of aseptic techniques prevents pure cultures from being contaminated.

A contaminated culture occurs when unknown microorganisms are inadvertently introduced often via poor technique. In addition, bacterial colony morphology (the shape of a group of bacteria) can help distinguish among different species when using a plate.

Question 24

Understand isolation techniques

Answer 24

• The streak plate is the primary mechanism for isolating bacteria. This technique utilizes a four quadrant dilution that systematically reduces bacterial numbers until they are diluted enough to form isolated colonies. The procedure is shown in Figure 5 and can be separated into four defined steps. Carefully read the following steps as you will be streaking a number of plates during this experiment.

Question 25

Know the steps needed to produce a streak plate

Answer 25

1. A bacterial sample must be collected. The bacterial sample may be collected by rubbing a sterile cotton swab over a test area, such as an elevator button or a place on the human body. Alternately, the bacterial sample may be obtained from a previous culture. Using aseptic methods, the sample is spread over a small portion of the plate, usually about ¼ of the plate size. This first inoculation may be referred to as the “first quadrant” and is shown in Figure 5A.
2. A small portion of the bacteria from the first quadrant is then spread to a second quadrant of the plate so as to isolate and “dilute” a portion of the microorganisms. An instrument called an inoculation loop is sterilized and then placed flat in the center edge of the first quadrant. The inoculation loop is used to spread a small portion of the organism from the first quadrant into the second quadrant, as shown in Figure 5B. Notice in the figure that it is only necessary to move into the first quadrant a few times. The second quadrant is created with a small amount of sample that is pulled out from the first quadrant. It is recommended that you work from the middle of the plate each time to maximize the amount of surface area used. Using the complete plate will optimize the dilution effect.
3. The microorganisms are again spread using the inoculation loop. After sterilizing, the loop is used again to spread a portion of the sample from quadrant two into a new, third quadrant of the plate. Again, it is recommended that you work from the middle of the plate to cover as
   much surface area as possible. See Figure 5C.
4. The microorganisms are spread into the final quadrant using the sterilized inoculation loop. Care must be taken to avoid accidently coming into contact with the other previously formed quadrants. If the loop accidentally comes in contact with the first and second quadrants, the dilution is lost. See Figure 6 for an example colonies growing on a streak plate.

Question 26

Understand why a streak plate is beneficial

Answer 26

a streak plate helps you to isolate bacteria into one colony instead of many

Question 27

Understand morphology of microorganisms

Answer 27

Morphology is the size, shape, and other physical characteristics that can be used to identify microorganisms. Even without a microscope, information about microbes can be acquired through observation on a plate. As shown in Figure 7, different microorganism colonies can have different sizes, shapes, and colors that can be seen on the plate. Additional morphological characteristics include whether the edge of the colony is smooth or rough and whether the centers of the colony are raised or indented. A certain species will always show this same morphology on a particular media. Attention to detail allows the creation of pure cultures and eventual identification of a microorganism. As you perform the experiment, pay very close attention and try to note the differences you see in the isolated colonies you create.

Question 28

Understand Escherichia coli, Saccharomyces cerevisiae, Staphylococcus epidermidis

Answer 28

Escherichia coli is a rod-shaped bacteria commonly found in the lower intestine of warm-blooded organisms. See Figure 8. Most E. coli strains are harmless and part of the normal flora of the gut. E. coli was one of the first organisms to have its genome sequenced and is a model organism for studies involving bacterial conjugation, genetics, and evolution.

Saccharomyces cerevisiae, also known as baker’s yeast, is an ovoid single-celled fungi found on ripe fruits in nature. See Figure 9. S. cerevisiae is harmless to most humans, but has been shown to irritate the digestive system of individuals suffering from Crohn’s disease and ulcerative colitis. S. cerevisiae is one of the most intensively studied eukaryotes and serves as a model organism for studies involving fermentation, proteins, genetics, and aging.

Staphylococcus epidermidis is a coccus-shaped bacteria commonly found on the skin of humans. See Figure 10. S. epidermidis is harmless to most individuals and considered part of the normal skin flora. S. epidermidis has been linked to infections in individuals with catheters or other
surgical implants. S. epidermidis is a model organism for the study of osmotic pressure regulation, antibiotic resistance, and biofilms.

Question 29

Understand experiment and the results observed

Answer 29

be able to define the morphology of S. cerevisiae and your skin sample
LOOK AT PICTURE ON SLIDES

Question 30

Understand safe handling of microbes

Answer 30

ASEPTIC TECHNIQUE

1. Hands must be washed, work surfaces must be cleared of unnecessary items, and sterilized with a 10% bleach solution or a bleach-based cleaner before beginning the Experimentation. See Figure 1.
2. The equipment used to isolate cultures has to be sterilized. Either an open flame or isopropyl alcohol may be used to eliminate contamination that is naturally picked up from the environment. In campus laboratories, equipment is often sterilized with an autoclave or micro-incinerator.
3. When opening cultures or sterile media:
   a. Work quickly and efficiently.
   b. Do not talk or breath over the culture.
   c. When tubes and vials are open, keep them tilted away from the mouth and nose.
   d. Never lay caps or lids on a bench or work area, as these areas most likely have contaminants.
   e. When working with a culture, ensure the instruments used are sterile. this will include inoculation loops and swabs. Sterile instruments are provided by Hands-On Labs, Inc. (HOL) You must work to maintain the instruments’ sterility
4. Some general precautions include the following:
   a. Avoid producing aerosols (anything that can be introduced into the air). This includes avoiding splashing when pipetting media and inoculating cultures.
   b. Keep the lid to the culture closed whenever possible.
   c. Incubate plates inverted (agar-side up). Agar contains a lot of water. When plates are incubated with the lid side up, water will condense on the lids; this condensation can drip back onto the agar plate and possibly contaminate and/or smear the results.
   d. Always write on the bottoms of the plates, not the lids. This will prevent unlabeled plates if lids are misplaced.
   e. When not in use, tubes and plates should be properly stored away from children and pets.
   f. All equipment and work surfaces should be cleaned with a 10% bleach solution or bleach based cleaning product at the end of the experiment.

Question 31

Know vocabulary listed of bacteria morphology

Answer 31

COCCUS - a spherical or round-shaped bacterium with a diameter of about 0.5 μm

BACILLUS - a cylindrical or rod-shaped bacterium about 0.5 μm to 20 μm in length

SPIRILLA - a helical or spiral-shaped bacterium about 15 μm in length

DIPLOCOCCI - a pair of cocci

TETRADS - group of four cocci

SARCINA - cocci can also form cubical packets of eight cells, sixteen cells, and even greater numbers of cells, which are all called

STAPHYLOCOCCI - Cocci can also aggregate into hundreds of cells into grape like clusters and irregular clumps called

DIPLOBACILLI - Bacilli have fewer arrangements but can pair together end-to-end forming

STREPTOBACILLI - link together forming a chain of cells

SIMPLE STAIN - One dye is used to directly stain the bacterial cell or the background. Cell shape, size, and arrangement may be determined with simple staining techniques.

DIRECT STAIN - which only color the cell’s cytoplasm and leave the background outside of the bacterial cell colorless. Examples of basic dyes are crystal violet, methylene blue, safranin, basic fuchsin, and malachite green.

NEGATIVE STAIN - - A type of simple stain; the cell is unstained and appears as a bright, often colorless object against a dark-stained environment.

DIFFERENTIAL STAIN - - Two or more dyes are used. Cell morphology can be described and identification of the structural components (composition of the cell wall and other cell features like flagella, capsules, or endospores) is possible. Figure 5 shows one type of differential stain called a Gram stain.

CHROMOPHORES - Stains are salts dissolved in a liquid and are composed of positive (+) and negative (-) ions. One of the ions is colored depending on the chemical composition of the solution. The colored ions are called chromophores. Chromophores have a positive or negative charge depending on whether the dye is basic (pH>7) or acidic (pH<7). The charge of the chromophores controls how the dye interacts with bacterial cells during the staining process.

BASIC STAIN - contains positively charged (+) chromophores. Because the cell membrane is slightly negative (-) in charge and opposite charges tend to attract, basic dyes (+) have a high affinity for the cell’s surface. Basic dyes (+) are used for direct stains which only color the cell’s cytoplasm and leave the background outside of the bacterial cell colorless. Examples of basic dyes are crystal violet, methylene blue, safranin, basic fuchsin, and malachite green.

ACIDIC STAIN - contains negatively charged (-) chromophores. Acidic dyes (-) are used for negative stains in which the dye is repelled from the cell’s negatively charged (-) surface. Negative stains color only the background or environment outside the cell. Congo red, india ink, and nigrosin are examples of acidic dyes.

FIXATION - During staining, a thin film of cells called a smear is applied to a blank microscope slide. Often the cells are preserved on the slide through a fixation process, whereby a flame is used to kill the bacteria and firmly affix the cells to the slide. It is important to note that heating causes the cell to shrink in size and some cell features may become distorted. Overheating can cause the cells to deteriorate, removing them from the surface. In negative staining, the bacteria are not affixed by heating. In fact, if the bacteria is still alive it can act as a potential contaminant or pathogen.

GRAM STAIN - uses two dyes to distinguish Gram-positive and Gram-negative bacteria. Like simple stains, differential stains can provide information on cell morphology such as shape, size, and arrangement.

GRAM-POSITIVE - Gram-positive cells have a thick outer cell wall composed of a mesh-like polymer called peptidoglycan, adjacent to an interior plasma membrane. See Figure 7. Teichoic acids are only found in the cell wall of Gram-positive bacteria. The teichoic acids are a group of molecules that run perpendicular to the peptidoglycan sheets and provide structural support. The size of the cell wall of Gram-positive cells ranges from 20 – 80 nm in thickness and is cross-linked in two dimensions making a strong, more rigid framework.

GRAM NEGATIVE - Gram-negative cells have a thin cell wall comprised of a single layer or sheet of peptidoglycan sandwiched between an outer and inner cell membrane. The thin cell wall is only cross-linked in one dimension and is flexible but more susceptible to lysis. The outer cell membrane also contains specialized molecules or proteins unique to Gram-negative cells called lipopolysaccharides (LPS) which are endotoxins that produce reactions such as fever or shock in Gram-negative infections. The size of the cell wall of Gram-negative cells ranges from 2 – 8 nm in thickness.

PEPTIDOGLYCAN - mesh-like polymer called peptidoglycan

TEICHOIC ACIDS - are only found in the cell wall of Gram-positive bacteria. The teichoic acids are a group of molecules that run perpendicular to the peptidoglycan sheets and provide structural support.

LIPOPOLYSACCHARIDES - outer cell membrane also contains specialized molecules or proteins unique to Gram-negative cells called lipopolysaccharides (LPS) which are endotoxins that produce reactions such as fever or shock in Gram-negative infections. The size of the cell wall of Gram-negative cells ranges from 2 – 8 nm in thickness

ENDOTOXINS - produce reactions such as fever or shock in Gram-negative infections

GRAM - VARIABLE - As Gram-positive cells age (24-48 hours old), their cell wall degrades causing the peptidoglycan layer to become weak and the crystal violet dye to leak and wash away during the decolorization step. These Gram-positive cells are known as Gram-variable cells and may appear Gram-negative or may appear as both types of cells when stained.

Question 32

Understand bacterial morphology (coccus, bacillus, spirilla)

Answer 32

• Coccus (cocci, plural) - a spherical or round-shaped bacterium with a diameter of about 0.5 μm
• Bacillus (bacilli, plural) - a cylindrical or rod-shaped bacterium about 0.5 μm to 20 μm in length
• Spirilla (spirillum, plural) - a helical or spiral-shaped bacterium about 15 μm in lengthFigure

Question 33

Understand how bacterial cells arrange together

Answer 33

Bacteria can exist as single cells or they may naturally group into colonies, clusters, or chains. Their arrangement is determined by the way the cells divide and attach to other cells in the colony. Thus, cells can be categorized according to their arrangement. Only cocci and bacilli form organized arrangements; spirillum remain as single cells and do not form clusters or chains.

Cocci have greater variation in their arrangement than bacilli. When a coccus bacterial cell dividesbut the daughter cells fail to fully separate after cell division, a pair of cocci is formed and the pair is called a diplococci. When cocci fail to separate and remain in groups of four, they form squares called tetrads. Cocci can also form cubical packets of eight cells, sixteen cells, and even greater numbers of cells, which are all called sarcina. Cocci can also aggregate into hundreds of cells in continuous, bead-like chains called streptococci and into grape like clusters and irregular clumps called staphylococci.

Bacilli have fewer arrangements but can pair together end-to-end forming diplobacilli or link together forming a chain of cells called streptobacilli. Bacilli that line up side-by-side at angles in an arrangement or in stacks are called palisades. See Figure 4 for examples of bacterial morphologies.

Question 34

Understand why staining of bacterial cells is needed

Answer 34

Stains, also called dyes, are often used to observe cell morphology with a microscope. Staining increases the contrast between the cells and their background. A variety of staining techniques exist, including simple stains (direct stains and negative stains) and differential stains.

Question 35

Understand each of the staining techniques

Answer 35

• Simple stain - One dye is used to directly stain the bacterial cell or the background. Cell shape, size, and arrangement may be determined with simple staining techniques.
• Direct stain - A type of simple stain; only the bacterial cell becomes colored, leaving the background or external environment clear and colorless.
• Negative stain - A type of simple stain; the cell is unstained and appears as a bright, often colorless object against a dark-stained environment.
• Differential stain - Two or more dyes are used. Cell morphology can be described and identification of the structural components (composition of the cell wall and other cell features like flagella, capsules, or endospores) is possible. Figure 5 shows one type of differential stain called a Gram stain.

Question 36

Understand what stains are and what chromophores are

Answer 36

Stains are salts dissolved in a liquid and are composed of positive (+) and negative (-) ions. One of the ions is colored depending on the chemical composition of the solution. The colored ions are called chromophores. Chromophores have a positive or negative charge depending on whether the dye is basic (pH>7) or acidic (pH<7). The charge of the chromophores controls how the dye interacts with bacterial cells during the staining process.

Question 37

Understand the different types of stains and how they interact with the cell

Answer 37

A basic stain contains positively charged (+) chromophores. Because the cell membrane is slightly negative (-) in charge and opposite charges tend to attract, basic dyes (+) have a high affinity for the cell’s surface. Basic dyes (+) are used for direct stains which only color the cell’s cytoplasm and leave the background outside of the bacterial cell colorless. Examples of basic dyes are crystal violet, methylene blue, safranin, basic fuchsin, and malachite green.

An acidic stain contains negatively charged (-) chromophores. Acidic dyes (-) are used for negative stains in which the dye is repelled from the cell’s negatively charged (-) surface. Negative stains color only the background or environment outside the cell. Congo red, india ink, and nigrosin are examples of acidic dyes. See Figure 6.

Question 38

Understand fixation

Answer 38

During staining, a thin film of cells called a smear is applied to a blank microscope slide. Often the cells are preserved on the slide through a fixation process, whereby a flame is used to kill the bacteria and firmly affix the cells to the slide. It is important to note that heating causes the cell to shrink in size and some cell features may become distorted. Overheating can cause the cells to deteriorate, removing them from the surface. In negative staining, the bacteria are not affixed by heating. In fact, if the bacteria is still alive it can act as a potential contaminant or pathogen.

Question 39

Understand Gram stain

Answer 39

All bacteria cells can be classified into two groups based on differences in the structure and composition of the cell wall. A widely used form of differential staining, the Gram stain, was developed in 1884 by the physician Hans Christian Gram. The Gram stain uses two dyes to distinguish Gram-positive and Gram-negative bacteria. Like simple stains, differential stains can provide information on cell morphology such as shape, size, and arrangement.

Cells that retain the primary stain (crystal violet) throughout the Gram stain procedure are Gram-positive cells. Gram-positive bacteria have cell walls that are thick and extensively crosslinked, allowing the dye complex to stay bound and fixed in the decolorization step. Cells that are colorless after the decolorization step but counterstain with a second dye (red safranin) are Gram-negative. Gram-negative bacteria have an outer cell membrane which is adjacent to and covers the cell wall and is composed of lipids or mostly water. During the decolorization step, the cell membrane is dissolved, exposing the thin cell wall and allowing the crystal violet dye to be washed away. Colorless Gram-negative cells are counterstained in the final step to increase contrast for visualization and identification.

Question 40

Understand the difference between Gram-positive and Gram-negative bacteria

Answer 40

Gram-positive cells have a thick outer cell wall composed of a mesh-like polymer called peptidoglycan, adjacent to an interior plasma membrane. See Figure 7. Teichoic acids are only found in the cell wall of Gram-positive bacteria. The teichoic acids are a group of molecules that run perpendicular to the peptidoglycan sheets and provide structural support. The size of the cell wall of Gram-positive cells ranges from 20 – 80 nm in thickness and is cross-linked in two dimensions making a strong, more rigid framework.

Gram-negative cells have a thin cell wall comprised of a single layer or sheet of peptidoglycan sandwiched between an outer and inner cell membrane. The thin cell wall is only cross-linked in one dimension and is flexible but more susceptible to lysis. The outer cell membrane also contains specialized molecules or proteins unique to Gram-negative cells called lipopolysaccharides (LPS) which are endotoxins that produce reactions such as fever or shock in Gram-negative infections. The size of the cell wall of Gram-negative cells ranges from 2 – 8 nm in thickness. See Figure 8.

Question 41

Know the Gram stain procedure and what you would observe at each stage

Answer 41

1. crystal violet dyes all bacterial cells, regardless of peptidoglycan thickness
2. Iodine reacts with crystal violet, forming a complex that allows dyes to better adhere to the bacterial cells
3. Decolorizer dehydrates the Gram-positive cell wall, trapping in dyes; decolorizer washes dyes from the Gram-negative bacteria
4. Safranin has no effect on Gram-positive bacteria; safranin causes Gram-negative bacteria to become pink

Question 42

Understand experiment and the results observed of staining techniques

Answer 42

Direct stain - it colors the bacteria and fixes them to the slide
Negative stain - can view cells alive and it colors the background
differential stain - saw both gram-negative and positives

Question 43

know vocabulary listed of enumeration, dilution, and plate counts

Answer 43

EXPONENTIAL GROWTH - growth of bacteria exponentially

BINARY FISSION - method of asexual reproduction during which a single cell (parent cell) will split into two cells (daughter cells). The process then continues as each of these cells divide into two more cells, thereby doubling the population size with each generation. During binary fission, cells continuously perform DNA synthesis and divide. See Graph 1. Prokaryotic cells, unlike eukaryotic cells, have no cell cycle with a specific time frame for DNA synthesis.

VIABLE PLATE COUNT - common method used to determine the number of organisms in a culture. It is assumed that each of the colonies arose from a single cell or a group of attached identical cells that were not separated during plating. The viable plate count can be accurate at both high and low bacterial concentrations. To perform this procedure, a series of dilutions must be created. Under normal circumstances, the number of bacteria in a sample would far exceed that which can be directly counted on a single plate.

COLONY FORMING UNIT - Samples are taken from the dilution series and spread over a plate. After an incubation period, the number of colonies that form are counted. Each colony on a plate is referred to as a colony forming unit or CFU. Each colony is the result of an isolated cell from the inoculation solution. Plates are considered viable when they can be used to accurately estimate the total numbers of microorganisms on a plate. A viable plate contains between 30-300 CFUs. CFU numbers less than 30 reduce accuracy when calculating the number of cells in the original sample. CFU numbers greater than 300 result in the merging of individual colonies.

Question 44

Understand binary fission and how microbes reproduce

Answer 44

Many microbes exhibit exponential growth through binary fission under ideal conditions. Binary fission is a method of asexual reproduction during which a single cell (parent cell) will split into two cells (daughter cells). The process then continues as each of these cells divide into two more cells, thereby doubling the population size with each generation. During binary fission, cells continuously perform DNA synthesis and divide. See Graph 1. Prokaryotic cells, unlike eukaryotic cells, have no cell cycle with a specific time frame for DNA synthesis.

Because many microbes exhibit exponential growth with uninhibited DNA synthesis and division, a few cells can reproduce into many cells over a short period of time. For example, if a single cell was to divide every 30 minutes, it would only require 10 hours to have a population greater than a half million cells. This is a contributing factor to the rapid spread of illness caused by microbes.

The small size and rate of reproduction of microbes prohibits an accurate determination of cell numbers within a sample. However, microbiologists do have techniques available for estimating population size. In this experiment, you will estimate the number of Saccharomyces cerevisiae present in a sample by performing a viable plate count from a series of dilutions.

Question 45

Understand exponential growth

Answer 45

rate of growth exponentially

Question 46

Understand what a viable plate count is and how it is used

Answer 46

The viable plate count is a common method used to determine the number of organisms in a culture. It is assumed that each of the colonies arose from a single cell or a group of attached identical cells that were not separated during plating. The viable plate count can be accurate at both high and low bacterial concentrations. To perform this procedure, a series of dilutions must be created. Under normal circumstances, the number of bacteria in a sample would far exceed that which can be directly counted on a single plate.

Question 47

Understand what a colony forming unit is

Answer 47

Samples are taken from the dilution series and spread over a plate. After an incubation period, the number of colonies that form are counted. Each colony on a plate is referred to as a colony forming unit or CFU. Each colony is the result of an isolated cell from the inoculation solution. Plates are considered viable when they can be used to accurately estimate the total numbers of microorganisms on a plate. A viable plate contains between 30-300 CFUs. CFU numbers less than 30 reduce accuracy when calculating the number of cells in the original sample. CFU numbers greater than 300 result in the merging of individual colonies.

Question 48

Understand dilution and how to calculate

Answer 48

To perform a viable plate count, basic rules of dilution must be used. Dilution requires the aseptic transfer of a known volume of a sample into a known volume of diluent. The following principles must be followed:

1. After diluting a solution, the number of cells in the original solution will always exceed the numbers in a diluted sample.
2. As long as the surface area of agar is the same, the total amount of agar used on a plate is not relevant unless it dilutes the sample.
3. The volume of the sample plated is relevant in the dilution calculations.
4. Any individual dilution is calculated using the following formula:
5. Successive dilutions are multiplied together to determine the final total dilution.
6. Only plates containing colonies within the 30-300 range are counted and recorded for accuracy.
7. To determine the number of CFUs in the original culture, the number of colonies on the viable plate is multiplied by the total dilution factor of that plate. For example, if it is determined that there are 57 CFUs on a plate that was diluted 1,000 fold, then the original culture would have contained 57,000 CFU/mL.
8. If there is more than one plate for a dilution, the CFUs of all the plates that have that dilution are averaged together prior to multiplying by the dilution factor.
9. If more than one dilution that falls within the 30-300 CFU range, only the least diluted plate is used in the calculations.

Dilution = Volume of sample added (mL) / Total Volume (mL of Sample + mL of Diluent)

Dilution = 1 mL / 1 mL + 9mL = 1:10 Dilution

Question 49

Know what level of CFU range is recorded for accuracy

Answer 49

30-300

Question 50

Understand how to perform a dilution including the calculations

Answer 50

Dilution = 1 mL / 1 mL + 9 mL = 1:10 dilution

Question 51

Understand experiment and the results observed of enumeration, dilution, and plate counts

Answer 51

pick the one between 30-300 and do the equation to find the answer